Project Summary/Abstract Bacterial pathogens are increasingly evolving drug resistance under natural selection from antibiotics in medicine, agriculture, and nature. Meanwhile, bacteria ubiquitously encounter bacteriophages and rapidly evolve phage resistance. However, the role of phages in driving drug resistance and sensitivity remains unclear. Phage selection can specifically interact with antibiotic selection in complex ways. For instance, the evolution of bacterial resistance to some phages increases resistance to antibiotics. On the other hand, some phages force bacteria into evolutionary tradeoffs between phage and antibiotic resistance. We have previously shown that phage which force such tradeoffs can drive the evolution of restored drug sensitivity in bacteria through the alteration of phage-targeted antibiotic efflux pumps. However, there are many non-efflux pump genes associated with drug resistance. For instance, Escherichia coli has 283 genes associated with drug resistance, only three of which are characterized efflux-related genes. It is unknown how phages interact with uncharacterized cryptic drug resistance-associated genes and the conditions where selection by phages that use drug-associated genes may restore drug sensitivity. To better understand the interactions of phage and antibiotic resistance, we will identify phages that use cryptic drug resistance genes and, importantly, we will identify conditions in which phage selection drives the evolution of increased drug sensitivity. Extending our previous work into a study system with excellent genetic tools, we will use microbiological, evolutionary, and molecular approaches to characterize interactions between our collection of phages and drug resistance associated genes in E. coli. This work focuses on the ecological and evolutionary mechanisms not addressed in previous studies. We will screen our E. coli phage collection (33 phages) against a collection of 283 E. coli gene knockouts to identify phages that specifically rely on drug-resistance-associated alleles. We will then test how microbial community dynamics are influenced by phages and in turn alter the evolution of antibiotic resistance, characterizing general and drug- related evolutionary tradeoffs in communities of increasing complexity. Finally, we will analyze drug-resistance- targeting phage through life cycle characterization and whole genome sequencing and annotation, generating well-characterized phages for future study. This proposal leverages existing molecular biology knowledge of E. coli to survey every gene associated with drug resistance and determine whether phages interact with those genes and how phage selection alters the evolution of drug sensitivity. Within microbial evolutionary biology, this project will reveal how microbial communities mediate and alter evolutionary tradeoffs. More broadly for medical microbiology, this work will uncover relationships between drug resistance and phage resistance, increasing our understanding of the mechanisms by which drug resistance ? and sensitivity ? can evolve at clinical timescales. Should lytic phage be broadly applied in medicine, our project fills fundamental knowledge gaps required for their effectiveness and sustainability.